Platelet promoting protein and the usage thereof

ABSTRACT

The present invention discloses a protein that has strong affinity to thrombopoietin receptor (C-MPL) and the nucleotide sequences of the protein. The protein is capable of increasing the numbers of platelets and enhancing the blood clotting in vivo and is named as platelet promoting protein (PPP). The protein and its nucleotide sequences can be used for the treatment of blood diseases including thrombocytopenia.

FIELD OF THE INVENTION

The present invention relates to a bio-medicament, and more specifically relates to a protein for increasing the numbers of platelets and its applications in the treatment of blood diseases.

BACKGROUND OF THE INVENTION

As an important component of blood, platelets are responsible for hemostasis in response to vascular injury and involved in the repairment of injured blood vessels. Low level of blood platelets can be life-threatening as it is prone to a mass loss of blood. At the present, platelet transfusion is a top choice for treatment for patients of thrombocytopenia. However, like other blood products, the platelets are short in shelf life, and are easy to be contaminated with blood pathogens such as hepatitis B virus and AIDS virus, and often elicit allergenic reactions among recipients.

Thrombopoietin (TPO) plays its role of growth factor for thrombopoiesis by binding to its receptor MPL, which is made up of three parts, MPL-EC(_(26-491aa)) (extracellular domain), transmembrane domain, and cytoplasmic domain. In 1994, five groups of scientists simultaneously cloned TPO. The successful cloning of TPO had injected new hopes and approaches for the treatment of thrombocytopenia. However, the clinical data indicated that the efficacy of TPO towards thrombocytopenia varied among patients. At the same time, the side effects of TPO were also observed. Two of the major side effects were: [1] TPO activated platelets, stimulated its aggregation, and thus lead to the formation of blood clotting; and [2] antibodies to TPO being generated after TPO administration (Archimbaud E, et al. Blood. 1999; 94:3694-3701; Schiffer C A, et al, Blood. 2000; 95:2530-2535; Nash R, et al. Blood. 1997; 90 suppl. 1:262a). The search for alternative therapeutic proteins or cytokines continues in the art.

The yeast two-hybrid system is a suitable system for the study of protein-protein interactions. The system, which contains two expression plasmids (plasmid A and plasmid B) as well as a yeast host, takes advantage of the fact that yeast transcription factors, such as LEXA or Gal4, comprises two separate domains: DNA-binding domain (DNA-BD) and transcription activating domain (AD). Plasmid A expresses a fusion protein of a bait protein and the DNA-BD; and Plasmid B expresses a fusion protein of a protein of interest and AD. After co-transformation of the two plasmids into the yeast host, the interaction of the bait protein and the protein of interest brings the DNA-BD and AD into close contact, which activates the transcription of the reporter gene. Therefore the system can be used to isolate ligands of bait proteins. The kits Matchmaker Two-Hybrid System 3, Matchmaker LexA Two-Hybrid System (Clontech) are commercially-available examples of the system.

SUMMARY OF THE INVENTION

The object of the current invention is to provide an isolated protein that has a function equivalent to the TPO.

The current invention also provides a nucleotide sequence for encoding the protein of the present invention and a plasmid that containing the DNA sequence.

Another object of the current invention is to provide the application of the protein, including using the protein as a medicament for treatment of a blood disease.

The current invention is carried out by using a yeast two-hybrid system, in particular, by using the extracellular domain of MPL (MPL-EC) as a bait protein to screen proteins in a human fetal liver cDNA library that interact with the MPL. The screening identified a protein that binds specifically to the extracellular domain of MPL. The protein of the present invention has 331 amino acids in length and has no homology to TPO in BLAST analysis. It is capable of stimulating the maturation of megakaryocytes and the formation of platelets and is consequently named as Platelet Promoting Protein, or PPP. The amino acid sequence of the PPP is shown as Sequence 2 in the Sequence Listing and its nucleic acid sequence is shown as Sequence 1 in Sequence Listing. The Platelet Promoting Protein or PPP of the present invention refers to the protein having the amino sequence as shown by Sequence 2 in the Sequence Listing.

Also provided by the invention are derivatives of the PPP. The derivatives of the “PPP” include: [1] mutants of the PPP, provided that the mutants retain the ability of increasing the numbers of platelets and enhancing the blood clotting in vivo; [2] variants of the PPP, which, as compared with the Sequence 2, comprise one or more conservative substitutions of amino acids; one or more deletions of amino acids; or one or more additions of amino acids; [3] a carboxyl terminal-truncated form or amino terminal-truncated form of the PPP having the Sequence 2; [4] a tandem repetition of partial or complete Sequence 2; and [5] a fusion protein of the PPP having the Sequence 2 and another protein or cytokine. One of such derivatives, for example, carries additional 2-6 histidines at the N-terminus of the Sequence 2.

The IUPAC nomenclature and symbolism for amino acid abbreviations was used in the present invention (European Journal of Biochemistry, 138:9-37, 1984).

To complete the invention, the inventors first amplified and isolated a 1.3 kb MPL-EC cDNA from the total human DNA using PCR primers MPLEC-F and MPLEC-R, which are complementary to the ends of the MPL-EC and contain appropriate restriction sites. The MPL-EC fragment was restricted and ligated into the polyclonal site of pLexA to generate a plasmid, named as pLexA-MPL-EC. The pLexA-MPL-EC and human fetal liver cDNA library were then co-transformed into Saccharomyces cerevisiae EGY48 and a positive clone was identified on an auxotrophic media and then DNA sequencing was conducted. The sequencing analysis of the positive clone revealed the clone had an insert having a nucleotide sequence shown as Sequence 1 in Sequence Listing. Its deduced amino acid sequence is given as Sequence 2 in Sequence Listing.

Insertion of PPP gene into expression vector pET-28b formed a expression vector, named as pET-PPP, which was subsequently transformed into E. coli BL21(DE3). The transformants were induced to produce His-PPP containing six continuous histidine residues at the N-terminus of the protein. The His-tag served as an affinity tag for the purification of PPP by using a cobalt-based immobilized metal affinity chromatography (Co²⁺IMAC) column.

The purified PPP was injected into normal mice and the amount of the circulating platelets was measured and the bleeding times were monitored. The results indicated that the His-PPP stimulated significantly the formation of platelets and increased the amount of platelets in the circulating blood.

The PPP of the invention is a potential medicament for the treatment of thrombocytopenia or/and hemorrhage. The protein of the present invention may be formulated into injections, powders, tablets, capsules, solutions, suspensions, or emulsions. The medicament may be administrated by oral administration or may be administered via subcutaneous injection, intravenous injection or intramuscular injection.

The present invention also provides a pharmaceutical composition comprising the PPP of the invention. The pharmaceutical composition may be prepared by mixing the PPP or the derivatives of the PPP that have the function of increasing the numbers of platelets, with pharmaceutically acceptable excipients. The excipients may be a liquid such as water, salines, phosphate buffers or albumin solutions; or a solid such as antioxidant agents, starches or dextrins. The pharmaceutical compositions are preferably to contain other hematopoietic growth factors such as interleukins, erythropoietins, macrophage colony stimulating factor (MCSF).

The PPP of present invention may be readily prepared in to various solutions by applying any known methods in the pharmaceutical field, such as by using a sterile saline, phosphate buffer and albumin solution. The concentration of the solution may range from 1 to 100 μg PPP per milliliter of the solution.

The PPP of the present invention may be administrated to patients in a dosage with the dosage of TPO as a reference, e.g. in the range of 1 to 1000 μg per kilogram of body weight per day. The dosage will be determined by a medically qualified physician, based on a variety of factors of the patients, including age, weight, severity of sickness, the cause and history of the disease.

The vectors and host cells described in the invention were obtained commercially. For example, pET-28b and E. coli BL21(DE3) were from Novagen, the yeast two hybrid system Matchmaker LexA Two-Hybrid System and Talon Metal Affinity Resin were from Clontech.

The current invention is further described with the figures and Examples. The invention is not limited by the detailed description provided in the Examples. Various modifications can be made by those skilled in the field and these modifications should be construed to fall within the scope of the invention defined by the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of plasmid pLexA-MPL-EC.

FIG. 2 shows the outline of plasmid pB42AD.

FIG. 3 shows the outline of expression vector pET-28b.

FIG. 4 shows the construction of the PPP expression vector pET-PPP.

FIG. 5 shows SDS-PAGE of His-PPP. The protein samples were separated on 10% SDS-PAGE and stained with Coomassie blue. His-PPP, with a size of 45 kDa, was indicated by the arrow. Lane 1 shows a protein size ladder; lanes 2 and 3 shows E. coli cell lysates before and after IPTG induction, respectively; lane 4 shows a soluble His-PPP crude extract; and lane 5 shows the His-PPP after cobalt affinity chromatography (Co²⁺IMAC) purification.

FIG. 6 shows the stimulation of platelet production by His-PPP in BALB/c mice. Normal BALB/c mice were subcutaneously injected with PBS (control, white column), or 10 μg/kg His-PPP (grey column), or 50 μg/kg His-PPP (black column) for 7 days. Twenty μL venous blood was collected from a small lateral cut in the tail vein on day 0, 4, 7, 10, 13, 16 and 19. The platelets were counted using an F-820 Sysmex electronic blood cell analyzer, and expressed as the mean±SD×10⁹/L.

FIG. 7 shows the reduction of bleeding time by His-PPP in BALB/c mice. Normal BALB/c mice were subcutaneously injected with PBS (control, white column) or 10 μg/kg His-PPP (black column) for 7 days. The bleeding times were measured on day 0, 4, 7, 10, 13, 16 and 19.

EXAMPLES

The examples presented below are for illustration of the invention only and are not intended to be regarded as the limitations of the invention. In the following examples, conventional practice or manufacturers' suggestion/protocol was followed in the cases where the conditions were not specified.

Example 1

Isolation of PPP that Interacts with the Extracellular Domain of MPL by Using a Yeast Two-Hybrid System

1.1 Construction of a Bait Protein Plasmid pLexA-MPL-EC

The primers MPLEC-F and MPLEC-R, with EcoRI and XhoI incorporated, were synthesized based on the sequence of MPL-EC as follows: MPLEC-F: 5′-CCGGAATTCCAAGATGTCTCCTTGCTGGCATCAGA-3′; MPLEC-R: 5′-CCGCTCGAGTTATCCGACCACGAGCTCCAGGG-3′∘

MPL-EC was PCR amplified with using the total human DNA as the template as indicated in FIG. 1. The PCR reaction mixture of a total 50 μl contained 1×PCR reaction buffer, 5 μM MPLEC-F, 0.5 μM MPLEC-R, 1 μg human total DNA, 2U Taq DNA polymerase (Fermentas), 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 1.5 mM MgCl₂. The PCR program used was: 94° C., 5 min; then 30 cycles of 94° C., 1.5 min, 55° C., 1 min, 72° C., 2 min; with an additional of 72° C., 10 min at the end of the program. The resultant PCR product of approximately 1,450 bps long was separated by and purified from 1% agarose gel, digested using EcoRI and XhoI, and ligated by using T4 ligase into pLexA (Clontech) to generate pLexA-MPL-EC (FIG. 1).

1.2 Identification of PPP

The screening system MATCHMAKER LexA Two-hybrid System (Clontech) is based on LexA and used for the detection of protein-protein interaction in the yeast (Gyuris et al., 1993). The detailed procedure was carried out as described by the protocol of the manufacturer.

The human fetal liver cDNA library in pB42AD (Clontech) was diluted and spreaded on LB plates and incubated overnight at 37° C. The cell colonies were collected by using sterile cotton tips and transferred into a LB broth and incubated overnight at 37° C. The plasmids were isolated by using E.Z.N.A.® Fastfilter Plasmid Miniprep Kit (Omega Bio-Tek). One hundred μg of pLexA-MPL-EC DNA from Example 1.1 and 100 μg human fetal liver cDNA library DNA were co-transformed into Saccharomyces cerevisiae EGY48 containing p80p-lacZ (Ura⁺, Lac⁺, Leu⁺); and the transformants were selected on SD/Gal/Raf/-His/-Trp/-Ura/-Leu auxotrophic medium containing X-Gal, prepared as suggested by the manufacturer. One positive clone of blue colony, named pB42AD-PPP, carrying an insert of approximate 1,300 bps was identified. The insert was sequenced and analyzed. The insert carried a complete coding region of 993 bps encoding a 331 amino acid peptide, as shown in Sequence 1 and Sequence 2. Blast analysis revealed that the sequence shares no homology with TPO but is identical to that of Human Nuclear Distribution Gene C (Matsumoto, N. and Ledbetter, D. H, Hum. Genet. 104, 498-504, 1999; Genbank database gi: 12052969). The protein was named as PPP, as it stimulates the formation of platelets (Example 3) and enhances the blood clotting function of platelets (Example 4).

Example 2 Construction of PPP Expression Vector pET-28b and the Expression and Purification of PPP

PPP was cloned into His-tag containing expression vector pET-28B (Novagen) (FIG. 3). The expressed protein His-PPP carried six continuous histidine residues at the N-terminus and can be purified by affinity chromatography.

pET-28b contains multiple cloning sites. The primes PPP-F and PPP-R were designed based on the restriction sites on the vector and the cDNA sequence of PPP: PPP-F: 5′-CGGGATCCGATGGGCGGAGAGCAGGAGGAGGA-3′, containing BamHI (underlined); PPP-R: 5′-CCGCTCGAGCTAGTTGAATTTAGCCTTGGAAA-3′, containing XhoI (underlined).

PPP DNA was amplified with using pB42AD-PPP DNA as the template. The PCR reaction mixture of a total 50 μl contained 1×PCR reaction buffer, 0.5 μM PPP-F, 0.5 μM PPP-R, 1 μg pB42AD-PPP DNA, 2U Taq DNA polymerase (Fermentas), 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 1.5 mM MgCl₂. The PCR program used was: 94° C., 5 min; then 30 cycles of 94° C., 1 min, 55° C., 1 min, 72° C., 1.5 min; and with an additional of 72° C., 10 min at the end of the program. The resultant PCR product of approximately 1,000 bps long was separated by and purified from 1% agarose gel, and digested by BamH1 and Xho1, and ligated into pET28b downstream and in frame with the His-tag to generate the construct pET-PPP (FIG. 4).

The pET-PPP was used to transform into E. coli strain BL21 (DE3), from Novagen. The transformant expressing His-PPP was grown in a LB medium containing 0.05 mg/ml kanamycin overnight at 37° C., and diluted 100 fold into the same medium and incubated at 37° C. to an A600 of 0.5-0.6, and induced with 0.5 mM IPTG for 4 h at 30 C. The bacterial cells were collected by centrifugation, suspended with a phosphate buffer of pH 7.4 containing 50 mM sodium phosphate, pH 7.4, 300 mM NaCl, and 1 mM PMSF, lysed by sonication and centrifuged at 10,000 g for 30 minutes to remove cell debris. The His-PPP containing supernatants was purified by using cobalt-based immobilized metal affinity chromatography (Co²⁺ IMAC) column (Clontech) as described by the protocol of the manufacturer to yield a soluble His-PPP of 45 kDa in size. The protein was more than 95% pure on 10% SDS-PAGE (FIG. 5).

Example 3 His-PPP Stimulates Platelet Formation in BALB/c Mice

The procedures of protein injection and venous platelet measurement were based on Kaushansky et al., Nature, Vol. 369, 1994, 565-568 with modifications.

The His-PPP purified as described at Example 2 was diluted into a stock solution of 10 μg/ml with PBS containing 0.1% BSA and used for the injection. Thirty normal male BALB/c mice of 6-7 week old were divided randomly into three groups of ten mice each. The first group was subcutaneously injected with 10 μg/kg His-PPP once per day for seven consecutive days; the second group was subcutaneously injected with 50 μg/kg His-PPP once per day for seven consecutive day; and the third group (control) was subcutaneously injected with PBS containing 100 μg/ml of BSA once per day for seven consecutive days. Twenty μl venous blood was collected from a small lateral cut in the tail vein on day 0, 4, 7, 10, 13, 16 and 19. The platelets were counted using of an F-820 Sysmex electronic blood cell analyzer (Sysmex Corp Ltd., Japan).

The results are shown in FIG. 6. At both dosages of His-PPP, significant increases (n=10, p<0.05) in platelet counts (expressed as mean±SD of platelets×10⁹/L) were observed on day 4 and 7. On day 4, 10 μg/kg group generated 15.8% more platelets (1346±54; n=10, p<0.05) and 50 μg/kg group generated 29.6% more platelets (1506±70; n=10, p<0.01) than the control (1169±72, n=10). On day 7, 10 μg/kg group generated 21.3% more platelets (1418±80; n=10, p<0.05) and 50 μg/kg group generated 27.4% more platelets (1489±57, n=10, p<0.05) than the control (1108±71, n=10). The platelet numbers of the both treatment groups gradually declined after the completion of the injections and returned to preinjection levels on day 19, 12 days after the end of the injection.

Example 4 His-PPP Reduces the Bleeding Time in BALB/c Mice

The bleeding time test measures the time taken for the blood flow, caused by incision of the mouse tail veins, to stop. The hemostasis test evaluates the blood clotting function of the platelets. The measurement was conducted as described by Alves-Rosa et al., Blood, Vol. 96, 2000, 2834-2840 with modifications.

The His-PPP purified as described at Example 2 was diluted into a stock solution of 10 μg/ml with PBS containing 0.1% BSA and used for the injection. Twenty BALB/c mice were divided randomly into two groups of 10 mice each. The first group was subcutaneously injected with 10 μg/kg His-PPP once per day for seven consecutive days; the second group (control) was subcutaneously injected with PBS containing 100 μg/ml of BSA once per day for seven consecutive days. The bleeding times were recorded on day 0, 4, 7, 10, 13, 16 and 19. The measurement was as follows: a wound of 20 mm wide was made using scissors at the tails of the mice and the blood was removed by gently contacting the cut site with paper filters every 30 seconds until there was no blood stain on the filter. The duration between the initiation and stoppage of the bleeding was recorded as the bleeding time.

The results are as FIG. 7 shows. On day 0, the bleeding times are almost identical for the two groups, both were around 5.5 minutes; on day 4, the bleeding time of His-PPP group was 4.2 minutes, 33.5% shorter than that of the control group (n=10, p<0.05) on day 7, the bleeding time of His-PPP group was 2.9 minutes, 50.8% shorter than that of the control group (n=10, p<0.05); and on day 10, the bleeding time of His-PPP group was 2.4 minutes, 61.9% shorter than that of the control group (n=10, p<0.05). After the injection ended, the bleeding time of His-PPP group started to bounce back gradually and returned to the pre-injection level on day 19, i.e., 12 days after the last injection.

The invention is not limited by the detailed description provided in the Examples. Various modifications can be made by those skilled in the field and these modifications should be regarded as within the scope of the claims of the invention. 

1. An isolated human-derived protein, having an amino acid sequence of: MGGEQEEERF DGMLLAMAQQ HEGGVQELVN TFFSFLRRKT DFFIGGEEGM AEKLITQTFS HHNQLAQKTR REKRARQEAE RREKAERAAR LAKEAKSETS GPQIKELTDE EAERLQLEID QKKDAENHEA QLKNGSLDSP GKQDTEEDEE EDEKDKGKLK PNLGNGADLP NYRWTQTLSE LDLAVPFCVN FRLKGKDMVV DIQRRHLRVG LKGQPAIIDG ELYNEVKVEE SSWLIEDGKV VTVHLEKINK MEWWSRLVSS DPEINTKKIN PENSKLSDLD SETRSMVEKM MYDQRQKSMG LPTSDEQKKQ EILKKFMDQH PEMDFSKAKF N;

or a derivative thereof with a function of increasing the numbers of blood platelets.
 2. The isolated protein according to claim 1, wherein the protein has additional 2-6 histidines at the N-terminus of the protein.
 3. The isolated protein according to claim 1 wherein the amino acid sequence of protein is encoded by the nucleotide sequence of: atgggcggagagcaggaggaggagcggttcgacggcatgttgctggccat ggctcagcagcacgagggcggcgtgcaggagcttgtgaacaccttcttca gcttccttcgacgcaaaacagactttttcattggaggagaagaagggatg gcagagaagcttatcacacagactttcagccaccacaatcagctggcaca gaagacccggcgggagaagagagcccggcaggaggccgagcggcgggaga aggcggagcgggcggccagactggccaaggaagccaagtcagagacctca gggccccagatcaaggagctaactgatgaagaggcagagaggctgcagct agagattgaccagaaaaaggatgcagagaatcatgaggcccagctcaaga acggcagccttgactccccagggaagcaggatactgaggaagatgaggag gaagatgagaaggacaaaggaaaactgaagcccaacctaggcaacggggc agacctgcccaattaccgctggacccagaccctgtcggagctggacctgg cggtccctttctgtgtgaacttccggctgaaagggaaggacatggtggtg gacatccagcggcggcacctccgggtggggctcaaggggcagccagcgat cattgatggggagctctacaatgaagtgaaggtggaggagagctcgtggc tcattgaggacggcaaggtggtgactgtgcatctggagaagatcaataag atggagtggtggagccgcttggtgtccagtgaccctgagatcaacaccaa gaagattaaccctgagaattccaagctgtcagacctggacagtgagactc gcagcatggtggaaaagatgatgtatgaccagcgacagaagtccatgggg ctgccaacttcagacgaacagaagaaacaggagattctgaagaagttcat ggatcaacatccggagatggatttttccaaggctaaattcaac.


4. A plasmid, comprising a nucleotide sequence for encoding the amino acid sequence of the protein according to claim
 1. 5. The plasmid according to claim 4, wherein the plasmid is the expression vector PET-PPP.
 6. A method of making a pharmaceutical composition comprising incorporating a protein according to claim 1 in medicament for treatment of thrombocytopenia or hemorrhage.
 7. The method according to claim 6, wherein the medicament is in a form of injections, powders, tablets, capsules, solutions, suspensions, or emulsions.
 8. The method according to claim 6, wherein the medicament is in a form for oral administration or in a form administration via subcutaneous injection, intravenous injection or intramuscular injection.
 9. A pharmaceutical composition, comprising the protein according to claim 1 or a derivative thereof with a function of increasing the numbers of platelets, and at least one pharmaceutically acceptable excipient.
 10. A method for treating a patient suffering from thrombocytopenia, comprising the step of administrating to the patient an effective amount of the protein according to claim 1 or having an effective amount of the protein according to claim 1 expressed in the patient. 